Transfer RNA (tRNA) is a small ribonucleic acid (RNA) molecule, generally about 74-95 nucleotides that has a key role in protein synthesis in the cytoplasm. The main function of tRNA is to bind to and transfer a specific activated amino acid to a growing peptide (protein) chain at the ribosomal site of protein synthesis during translation. tRNA molecule has a 3′ terminal site for the covalent linkage of a specific amino acid. The covalent linkage is catalyzed by an enzyme named aminoacyl tRNA synthetase. In addition, the tRNA molecule includes a three base region named the anticodon, which may base pair to the corresponding three base codon region on the mRNA molecule, which is the template for the protein synthesis. Each type of tRNA molecule may be attached to only one type of amino acid, however, since the genetic code includes multiple codons that specify the same amino acid, tRNA molecules bearing different anticodons may carry the same amino acid. (Lodish H, Berk A, Matsudaira P, Kaiser C A, Krieger M, Scott M P, Zipursky S L, Darnell J. (2004). Molecular Biology of the Cell. WH Freeman: New York, N.Y. 5th ed).
tRNA molecules are made and processed in the cell nucleus by a process known as transcription, while its site of action is in the cytoplasm, when participating in protein synthesis, as mentioned above. Hence, tRNA molecules translocate (move) between different subcellular locations. The dynamic movement and steady state accumulation of tRNAs in and between various subcellular locations may be dictated by the sensing of physiological states of the cell and may be regulated by mechanisms that are related to tRNA biosynthesis, function and turnover. Regulation of tRNA availability is central in the cellular response for the need of protein synthesis. Various cellular cues may dictate the function of tRNA by regulating the association of the tRNA with various cellular elements, such as, for example, association of tRNAs with amino-acyl-tRNA synthetases, association of tRNA with translation factors, association of tRNA with cytoplasmic polysomes, association of tRNA with ER-associated polysomes, and the like. In addition, alterations to the intracellular distribution of t-RNAs and sites of protein synthesis occur according to the onset of cellular programs such as cellular growth, division, differentiation, movement and cell-pathogen interactions. For example, viruses which generate localized foci of replication and assembly, termed viroplasms or viral factories, tailor the intracellular milieu to their needs, and may monopolize and concentrate the protein synthesis machinery in sites which differ significantly to those present in uninfected cells. (Castello, A., A. Quintas, et al. (2009), PLoS Pathog 5(8): e1000562; Katsafanas, G. C. and B. Moss (2007), Cell Host Microbe 2(4): 221-8; Qin, Q., C. Hastings, et al. (2009), J Virol 83(21): 11090-101; Smith, J. A., S. C. Schmechel, et al. (2006), J Virol 80(4): 2019-33).
It has previously been shown that in yeast, the tRNA retrograde process (the move/transport of tRNA from the cytoplasm to the nucleus), is energy-dependent, rapid, reversible, and may be responsive to nutrient availability. The re-export to the cytoplasm requires tRNA aminoacylation in the nucleus and probably the binding of eEF1A, which also is present in the nucleus (reviewed by Hopper A K, Pai D A, Engelke D R., FEBS Lett. 2010 Jan. 21; 584(2):310-7). Another publication (Shaheen H H, Horetsky R L, Kimball S R, Murthi A, Jefferson L S, Hopper A K. Proc Natl Acad Sci USA. 2007 May 22; 104(21):8845-50) has shown a retrograde tRNA transport in rat hepatoma cells upon amino acid starvation. Additional publication has shown that Lenti retroviruses use the tRNA retrograde cellular process to deliver their reverse-transcribed genome into the nucleus in non-dividing neuronal cells (Zaitvesa L., Mayers R., Fassati A. (2006), PLos Biol 4:e332).
Nevertheless, the retrograde transfer of tRNA to the nucleus has not been imaged in real time, while it is occurring, in viable cells. Moreover, a correlation between various cellular stress conditions and the changes (spatial and/or temporal) of tRNA subcellular localization have not been demonstrated in living cells. There is thus an ongoing need for methods that provide detection and measurement of the retrograde movement of tRNA in cells. There is further an ongoing need for methods that provide detection and measurement of the retrograde movement of tRNA in cells in real time. There is also a need for methods for detecting, measuring and/or assessing various cellular stress conditions in real time in viable cells, by tracking the subcellular tRNA localization and changes in said localization.